[Background] High-temperature stacking is a key step among the whole sesame-flavor Baijiu fermentation process, and vital for the formation of typical flavor of sesame-flavor Baijiu. [Objective] This study aimed to analyze prokaryotic and eukaryotic microbial communities during the high-temperature stacking process. [Methods] Illumina high-throughput sequencing was performed on the bacterial 16S rDNA V3–V4 region and the fungal ITS2 region of the high-temperature stacked Jiupei samples. The change pattern of prokaryotic and eukaryotic microbial community structure were analyzed. The changes in temperature, moisture, acidity, starch and reduced-sugar during stacking were also analyzed. [Results] At the beginning of 0–6 hours, the dominant bacteria were Bacillus, Weissella, and Acetobacter. After 20 h of stacking, the flora changed significantly. Weissella decreased and the Acetobacter increased. At the beginning, the dominant fungi were Aspergillus, Saccharomyces, and Pichia. After 6 hours, the proportion of Aspergillus dropped sharply, while Pichia and Saccharomyces gradually increased, and their absolute dominance maintained until the end of the stacking. The overall structure of the microbial community was temporarily affected by the turn-over operation (when it was piled up for 20 h), eventually returning to equilibrium. After 36 h of stacking, both prokaryotic and eukaryotic microbial community stabilized. Reduced-sugar data showed that the saccharification was rapid in the early stage, later became stable after 36 h. Temperature, acidity and starch content were also stable at this stage. [Conclusion] During the high-temperature stacking-fermentation process, fluctuation patterns of the prokaryotic and eukaryotic microbial community structure were different. The prokaryotic community changes were based on the turn-up at 20 h. The dominant species before the turn-up were Bacillus, Weissella and Acetobacte, the dominant strains after the turn-up were Weissella and Acetobacter, the latter was absolute dominant. Compared to prokaryotic community changes, the eukaryotic microbial community concentration gradually, by the end of the stacking, Pichia and Saccharomyces accounted for more than 93% of the total eukaryotic microbial. During the high-temperature stacking of the sesame-flavor Baijiu, the propagation of various prokaryotic/eukaryotic microbes and the corresponding changes of microbial communities laid the microbial basis for later high-temperature fermentation.